New 6-aminopenicillanic acid derivative

ABSTRACT

Penicillin derivative substituted at the 6 position with a (D)((2-amino-2-carboxy)-ethylthio)-acetamido group is obtained from Acremonium chrysogenum new strain ATCC 20 389 or by semisynthetic route. The product is an antibacterial agent.

United States Patent Troonen et al.

NEW 6-AMINOPENICILLANIC ACID DERIVATIVE Inventors: Hugo Troonen,Hoeilaart; Piet Roelants, Watermaal-Bosvoorde; Bernard Boon, Ohain, allof Belgium Assignee: Recherche et lndustrle Therapeutiques (R.I.T.),Genual, Belgium Filed: Mar. 7. 1974 Appl. No.: 449,180

U.S. Cl. 260/239.i: l95/260; 424/271 Int. Cl C07d 27/52 Field of Search260/239,]

References Cited UNITED STATES PATENTS 6/1963 Hobbs 260/2391 [451 May13,1975

3.761.466 9/!973 Bright 260/239.l

Primary Examiner-Leon Zitver Assistant Examiner-Gerald A. SchwartzAttorney, Agent, or Firm-Alan D. Lourie; William H. Edgerton 3 Claims,No Drawings 1 NEW 6-AMINOPENICILLANIC ACID DERIVATIVE HOOC S Cl-l 3 Ncoon and to a method for the production thereof.

Antibiotic RIT D-22l4 is an amphoteric compound susceptible to formsalts with pharmaceutically acceptable cations such as sodium potassium,calcium and ammonium and acids, said salts are preferably monosalts withcations e.g. the mono-saline, mono-potassium and mono ammonium salts.

According to this invention, antibiotic RIT D-22l4 is produced either bycultivating under controlled conditions a hitherto undescribed strain ofAcremonium chrysugenum species which has been deposited at the AmericanType Culture Collection (Rockville, Md., USA.) where it received theAcremonium chrysogenum ATCC 20 389 designation or by semi-syntheticroute.

For producing antibiotic RIT D-22l4, the Acremonium chrysogenum strainATCC 20 389 is cultivated in a culture medium containing assimilablesources of carbon, nitrogen, inorganic salts andL-carboxymethylcysteine, under submerged aerobic conditions until asubstantial amount of said compound is produced by said organism in saidculture medium, said culture medium being maintained at a temperature offrom approximately 20C to approximately 37C and the growth of theorganism being carried out for a period of approximately 36 to 96 hrsand the antibiotic RIT D-22l4 is recovered from said medium.

Acremonium chrysugenum ATCC 20 389 has been obtained by mutagenesis ofthe Cephalosporium sp. BROTZU strain (ATCC II 550).

We have found that the Acremonium chrysogenum ATCC 20 389 strain isauxotrophic for lysine and alpha-aminoadipic acid and its fermentationresults in the production of a number of antibiotic substances, e.g.penicillin N, Cephalosporin C, Cephalosporin P and/or antibiotic RITD-22l4, depending on the nature of the precursor incorporated to theculture medium.

The characteristics of the Acremonium chrysogenum ATCC 20 389 strain aregiven below and with reference to its parental strain ATCC II 550 and tothe Cephalospvrium chrysogenum ATCC l4 6l5 type strain.

The strains were examined for homogeneity and stability of theircharacters through cultures of one hurldred single-conidium isolates.The Brotzu strain showed good homogeneity in the colony characters onseveral culture media, while the RIT 02/6706 mutant revealed somevariation in the external pattern of the colonies. The type strain ATCCI4 615 was homogenous in 96 of the 100 single-conidium isolates, thefour remaining ones being not pigmented and morphologically distinct.

Each strain was compared by cultures of one massconidium and threesingle-conidium transfers on seven distinct culture media.

The formulae of the culture media are as follows: MYA4 malt extract 40g, yeast extract 3g, agar 20g per liter MYA2 malt extract 20 g, yeastextract 3 g, agar 20 g per liter PDA glucose 20 g, potato extract from200 g pelled potato in water, agar 20 g per liter CZA saccharose 30 g,sodium nitrate 3 g, dipotas sium hydrogen orthophosphate l g, magnesiumsulphate crist. 0.5 g, potassium chloride 0.5 g, ferrous sulphate 0.0lagar l5 g per liter YDPA glucose 20 g, yeast extract 5 g, bactopeptone10 g, agar 20 g per liter DYAA glucose 10 g, yeast extract 3 g,asparagine 0.5

g, dipotassium hydrogen orthophosphate l g, magnesium sulphate crist.0.5 g, hydrated ferric chloride 0.01 g, agar 25 g per liter WAKS glucosel0 g, beef extract 5 g, bactopeptone 5 g, sodium chloride 5 g, agar 20 gper liter Denotations of color are made by reference to their ref erencenumber in Seguy, E. Code Universe] des Cou leurs. Ed. Lechevalier,Paris, 1936.

SPECIES IDENTIFICATION Both parental and mutant strains exhibitmacroscopic and microscopic characters identical to those of theCephalosporium chrysogenum Thirum. and Sukap. type strain and arerepresenting this species.

The generic name Cephalosporium having been shown erroneous for thegenus (see W Gams in Cephalosporiumartige Schimmelpilze. Stuttgart1971), the earliest name Acremonium has been adopted and therefore theAcrenomiun chrysogenum designation is proposed for the strain RIT02/6706.

DESCRIPTION OF STRAIN ATCC 20 389 Culture in vitro, on malt extractyeast agar at 4 (MYA4).

Colonies, on MYA4, growing slowly and restrictedly (8-l0 mm after l0days at 22C), first white becoming ivory to rose-ivory (paler than S.l),reverse un colored to pale ochre (5.200) with no pigmentation of theculture medium, round shaped to angular, margin abrupt and crenate,surface elevated, strongly and densely wrinkled, major wrinkles high,radiate, irregularly undulate to sigmoid, sometimes furcate, anastomosedtransversely by lower minor wrinkles, thick, becoming larger andconvoluted and finally blending together into high mesanteriform massa,crateriform at the center, texture soft becoming ceraceous.

Hyphae mucilaginous, prostrate (aerial only on particular media),regular and cylindrical, narrow l-2.4 um diam., septate, perpendicularlybranched, with smooth and thin hyaline wall, aggregated in prostrateradiate and transverse ropes, often anastomosed with adjacent hyphae byshort unseptate anastomoses, breaking down at maturity into separatedhyphal cells of various shapes.

Conidiophores: phialides numerous arising solitary, lateral andperpendicular on the prostrate hyphae. erected, more or less undulate,simple, not branched, 25-60 14m length, 1.5-2.5 urn wide at the base,progressively attenuating towards the apex to l-l.5 um wide in acylindrical tube without collarette, always with one basal septum at 2-410) am above the hypha,

delimitating a basal cell including an hyphal portion and notdifferentiated, sometimes with a second septum at 2l0 pm from the basalone, with smooth and thin wall.

Conidia: phialospores produced enodgenously and singly at the apex ofthe phialide, agglutinating into a sphaerical dropplet of mucilage,elliptical or lanceolate, often attenuated and flattened at the base,larger at the lower half, often slightly strangulated at the mid dle,blunted or slightly pointed at the apical end, often asymmetrical inshape, amygdaliform or planoconvex, straight or Slightly curved, withsmooth thin mucilagi nous wall and often biguttulate contents, orvariable lenght, measuring 2.5ll Xl,53.5 um, mostly 4-7 X 2-3 am.

Hyphal cells resulting from the fragmentation of mature hyphae withswellings, thickened walls and gelatinized transverse septa, observedafter six weeks, with distinct shapes (1) linear, straight, cylindricaland (2) vermiform, undulate or contorted, cylindrical, not inflated,7-l0 X l-2 ptm, not numerous; (3) moniliform, simple or furcate,slightly inflated at intervals and the ends, not septate, ll5 X 25 um,numerous; (4) pan duriform fiddle-shaped, first short hyphal cellsswollen at both ends, strangulate at the middle, entire or septate, 5-10X 2-3 pm, becoming larger through unequal or equal swelling of each halfinto chlamidosporelike cells with thick wall, 54) um diam, numerous; (5)sphaeropedunculate, developed from the swelling of a single end of anhyphal cell, the other end remaining almost unchanged, swollen endglobose, thick-walled, 5-6 am diam., appendages cylindrical, l.52.5 umwide, numerous; (6) inflated hyphal cells, intercally or terminal, theswelling involving the entire lenght of the cell, subglobose to ovate,wall slightly thickened, 38 um, unfrequent.

On most of the used culture media, the strain ATCC shows absence ofyellow pigmentation, except a faint yellow tinge on MYAZ. Its growth isrestricted, submerged and dendroid on PDA and very reduced on CZA,because of the deficiency in lysine.

COMPARISON BETWEEN RIT 02/6706 MUTANT STRAIN AND PARENTAL STRAIN ATCC II550 The comparison with the parental strain makes possible thediagnostic differentiation of the mutant RIT 02/6706 on the base of thefollowing characters: growth, color of the surface and the reverse ofthe colony, diffusion of yellow pigment in the culture media, surfacepattern of the colony and the micromorphology and frequency of thedesintegrated hyphal cells in mature culture.

Some variation in the colony pattern is observed be tweensingle-conidium isolates of the RIT 02.6706 mutant, in the enlargmentand folding of the wrinkles up to their confluency into mcsenteriformmassa from the center up to the margin; but it represents differentstages of development of a common character.

No significant difference is observed in the shape and size of theconidiophores and conidia between the parental and mutant strains. Inboth strains also, sporulation occurs on the same culture media as DYAA,YDPA, WAKS and also CZA (if added with lysine for the mutant), and noton MYA4 or PDA.

COMPARISON BETWEEN ATCC 20 389 AND ITS PARENTAL STRAIN ATCC l l 550 ONDIFFERENT CULTURE MEDIA, AFTER 6 WEEKS AT 22 C.

I, On MYA4, Malt-yeast-agar 4 ATCC 20 389 Growth [6 mm. diam.

Color white to rose-ivory (paler than 8.190), re

verse pale ochre (S200); no pigment.

Surface wrinkles dense, thick, high, radiating, undulating, anastomosingin the depth, confluent at the center into a high mesenteriform andcrateriform massa, also sometimes confluent up to the margin.

Margin high abrupt and crenate.

Hyphal cells moniliform and vermiform up to 25 um widesphaeropedunculate and panduriform small, thickwalled, inflated up to 6pm diam; terminal inflated cells unfrequent up to 8 pm diam.

ATCC ll 550 Growth 2022 mm. diam.

Color soon intense yellow (5.272); reverse orange yellow (S256);diffusion of intense yellow pigment (5.242).

Surface wrinkles lower, radiating but zig-zag and furcate, developing areticulum by anastomose up to the margin, not confluent, with centeralmost not elevated.

Margin abrupt, crenate.

Hyphal cells linear and vermiform; sphaeropedunculate and panduriformlarger, up to 12 pm with thick wall.

2. On MYAZ, Malt-yeast-agar 2 ATCC 20 389 Growth 33 mm diam.

Color ivory to pale olive yellow (8.264); reverse pale yellowish (S228diffusion of yellow pigment none or very fainted.

Surface wrinkles undulate, radiating from the crateriform elevatedcenter, progressively attenuated to the margin, very poorly anastomosed.

Margin plane, flhrillose, submerged.

Hyphal cells mostly linear, vermiform and panduri form, often septate,5--l 5 X 235 pm; inflated cells up to 5 pm diam. unfrequent.

ATCC I1 550 Growth 40-50 mm. diam.

Color pale yellow (5259-260): reverse deep orange yellow (S226)diffusion of yellow pigment intense. Surface wrinkles low, poorlydeveloped from the center, radiate and undulate, more developed at themargin with anastomoses,

Margin extending under the agar.

Hyphal cells, mostly linear, doliform, toruloid, 5-15 X 2-3.5 ,um,panduriform septate 8l0 X 3-4 um, glohose or subglobose cells up to 6 umdiam.

3. on PDA, Potato-dcxtrose-agar.

ATCC 20 389 Growth 20 mm diam.

Color milky, reverse uncolored; diffusion of pigment none.

Surface almost entirely submerged, with local superficial white tufts.

Margin submerged, deeply dendroid.

Hyphal cells, mostly moniliform and panduriform,

much enlarged, thick-walled, -18 X 4-6 $1.; inflated cells intercalaryand terminal, elliptical or pyriform, up to 8 pm diam.

ATCC 11 550 Growth 37 mm diam.

Color: intense pure yellow (S271 reverse dark yellow (8.212); diffusionof yellow pigment intense. Surface wrinkled radiately at the centeronly, plane towards the margin.

Margin outlined finely dendroid.

Hyphal cells linear and moniliform 5-18 X 1.5-2.5 urn; panduriformirregular, thick walled, 10-14 X 3-5 am; few globose inflated cells upto 12 um diam.

4. on CZA, Czapeck agar.

ATCC 389 Growth 5-10 mm. very restricted (because of lysine deficiency).

Color milky, no pigment,

Surface submerged.

Margin dendroid.

Hyphae toruloid, 3-5 pm diam, with production of lateral globose cellsup to 4 pm diam., and intercalary panduriform cells up to 7 pm diam.

ATCC 11 550 Growth 45-50 mm.

Color dark yellow (5.258), reverse concolorous;

diffusion of yellow pigment intense.

Surface wrinkles regularly radiating from the center, furcate andattenuate at the margin.

Margin, regular, submerged.

Hyphal cells regular with some intercalary globose, elliptical, pyriformand panduriform cells, 5-15 X 3-5 am; some subglobose cells up to 10 umdiam.

5. on YDPA, Yeast-dextrose-peptone-agar.

ATCC 20 389 Growth 30 mm diam.

Color: pale ochre-rose (5.200) to light grey (8.235);

reverse amber; no yellow pigment,

Surface, on 20 mm diam., wrinkles thick, radiate from a center,undulate, bifurcate and attenuate near the margin, not anastomosed.

Margin diffuse, submerged, 5 mm wide.

Hyphal cells globose inflated cells up to 8 u diam.;

panduriform up to 12 X 4 pm.

ATCC 11 550 Growth 38 mm.

Color ivory turning pale yellow (5.319); some diffusion of pigment.

Surface, on 26 mm diam., with wrinkles radiate from not prominentcenter, sigmoid, attenuate at the margin, not anastomosed.

Margin diffuse, submerged, 5-6 mm wide.

Hyphal cells globose inflated up to 7 pm diam. or

doliform up to 10 X 4 am, intercalary.

6. on DYAA, dextrose-yeast-asparagin-agar.

ATCC 2038) Growth mm. Color rose-ivory (paler than 8.200), no yellowpigment.

Surface, on 15 mm diam, wrinkles fine radiate, straight, attenuate andbifurcate near the margin, not anastomosed.

Margin diffuse, submerged,'5 mm wide.

Hyphal cells intercalary inflated globose up to 8 am diam. or ellipticalup to 13 X 5 am.

ATCC 11 550 Growth 38 mm diam.

Color ivory turning pale yellow (S259); some diffusion of yellowpigment.

Surface, on 12 mm. diam, with wrinkles low, radiate,

no anastomosed.

Margin diffuse, submerged, 13 mm wide.

Hyphal cells some panduriform 10 X 3 pm; terminal and intercalaryinflated globose up to 6 pm diam. or elliptical up to 10 X 5 am.

7. on WAKS, Waksman's medium.

ATCC 20 389 Growth 25 mm.

Color rose-ivory (S250), reverse uncolored; no yellow pigment.

Surface, on 12 mm. diam., wrinkles few, reticulate not radiate,anastomosed and edging the surface.

Margin submerged 6-10 mm.

Hyphal cells linear; intercalary inflated globose up to 8 am diam. orelliptical up to 13 X 5 am; mostly panduriform up to 12 X 4 pm.

ATCC 11 550 Growth 50 mm.

Color: ivory turning pale yellow (3.319); some diffusion of yellowpigment.

Surface, on 25 mm diam, wrinkles radiate, sigmoid,

attenuate near the margin, not anastomosed.

Margin diffuse submerged, 5 mm wide.

Hyphal cells: terminal and intercalary inflated cells globose up to 7 pmdiam. or elliptical up to 10 X As indicated hereinabove, antibiotic RITD-2214 is produced by the cultivation of the Acremrmium chrysogenumstrain ATCC 20 389, Different culture media may be used for producingantibiotic RIT D-22l4 by cultivation of strain ATCC 20 389. The carbonsource may be for example dextrose, sucrose, maltose, dextrin, lactose,starch, vegetable oils and other carbon sources known to the art. Thenitrogen source may be for example casein hydrolysates, malt extract,fish meal, soybean meal, peanut meal, meat meal, corn steep liquor,peptones, amino acids or their analogues. As assimilable nitrogensources ammonium salts such as ammonium acetate, phosphate or sulphatemay also be employed. Minor elements necessary for optimum growth anddevelopment of the organism used for the production of antibiotic RITD-22l4 may also be included in the culture medium. Such trace elementscommonly occur as impurities in the other constituents of the medium inamounts sufficient to meet the growth requirement of the organismemployed in this invention.

Owing to the auxotrophic nature of the strain ATCC 20 389 it isessential to adequately supplement the medium with L(+) lysine foroptimum growth and development of the strain and with L(+)carboxymethyleysteine as a precursor for the production of antibioticRIT D-2214. Lysine and carboxymethylcysteine are preferably added so asto be present in a concentration of about 0.001 percent to 1 percentweight in volume of the total culture medium and preferably in a concentration of about 0.05 to 0.1 percent.

The culture medium may also contain those specific nutrients known tothe art for improving the production yield of cephalosporin C by aCephalosporium strain. Thus. the culture medium composition for theproduction of antibiotic RlT D-22l4 may be improved by adding either D.l. or DL methionine. L-cysteine. oleic acid. methyl oleate. ammoniumsulfate, calcium sulfate or others. Among them. DL-methionine is aparticularly valuable nutrient for improving the production yield ofantibiotic RlT D-22l4. DL-methionine may be supplemented to the mediumat a concentration from about 0.01 to 1 percent (weight/volume).optimally at about 0.2 to 0.5 percent.

The intial pH of the culture medium can be varied.

However. it has been found desirable that the initial pH of the mediumbe between 6.5 and 7.5. Submerged, aerobic culture conditions areconditions of choice for the production of antibiotic RlT D-22l4. Forpreparation of relatively small amounts. shake flasks and surfaceculture in bottles can be employed but for the preparation of largeamounts. submerged aerobic culture in sterile tanks is preferred. Themedium in the sterile tank can be inoculated with a sporulated suspension', but because of the growing lag experienced when a sporulatedsuspension is used as the inoculum, the vegetative form of the cultureis preferred. By thus avoiding the growth lag. more efficient use of thefor mcntation equipment is realized. Accordingly, it is desirable firstto produce a vegetative inoculum of the organism by inoculating arelatively small quantity of culture medium with the spore form of theorganism; and when a young active vegetative inoculum has been obtained.to transfer the vegetative inoculum aseptically to the large tank. Themedium in which the vegetative inoculum is produced can be either thesame as or different from the medium utilized for the large scaleproduction of antibiotic RlT D-22l4.

The organism which produces antibiotic RlT D-22l4 does grow over a widetemperature range between 2235 C.

Optimal production of antibiotic RlT D-22l4 seems to occur attemperature of 223() C. In general. maximum production of the antibioticoccurs within about 3 to 5 days after inoculation of the culture medium.

As is customary in aerobic. submerged culture processes. sterile air isblown through the culture medium. For efficient growth of the organismand antibiotic RlT D2214 production. the volume of air employed in theproduction tank is from 0.25 to volume of air per minute per volume ofculture.

The preferred volume is 054.0 volume of air per minute per volume ofculture medium.

The concentration of antibiotic activity in the culture medium can befollowed readily during the fermentation period by testing samples ofthe culture medium for their inhibitory activity against the growth oforganisms known to be inhibited by the presence of antibiotic RlTD-22l4.

The organism Alruligener viscuiurvis has been found particularly usefullfor this purpose owing to its very high sensitivity towards antibioticRlT 0-2214 and its lack of sensitivity towards the ccphalosporins Pgroup.

The testing of samples can be carried out by the well knownagar-diffusion method on Petri dishes or on plates.

The antibiotic activity produced during the fermentation occurs in theantibiotic broth. Accordingly. isolation techniques employed in theproduction of antibi otic RlT D-22l4 are designed to permit maximumrecovery of the antibiotic from the broth. Thus. for example. myccliumand undissolved solids are removed from the fermentation broth byconventional means such as filtration or centrifugation. and antibioticRIT D-22l4 can be recovered from the filtered or centrifuged broth byemploying extraction or adsorption techniques.

For the recovery of antibiotic RlT D-22l4 by adsorption techniques.various adsorbents and ion exchange resins can be used. for example.carbon. silica gel, alumina. cellulose, ion exchange resins andnon-ionic res ins. Antibiotic RlT D-22l4 can be adsorbed from an aqueoussolution onto one of the above or similar adsorbents. The adsorbedantibiotic RlT D-22l4 can be eluted from the adsorbent by suitableelution techniques, such as by washing the adsorbent on which theantibiotic RlT D-22l4 is adsorbed with a solvent. When the elution iscarried out by washing the ionic resins on which the antibiotic RlTD-22l4 is adsorbed. with a solution of. eg. ammonium formate, sodium acetate or potassium acetate. the process results in elu tion ofantibiotic RlT D-22l4 as the monoammonium. monosodium or monopotassiumsalt. respectively. When the elution is carried out by washing thenonionic resins on which the antibiotic RlT D-22l4 is adsorbed. theeluent is water.

It has been found that adsorbents previously described as suitable forthe adsorption of cephalosporin C and penicillin N. such as weakly andstrongly basic resins, for example the Amberlite resins of the lRA-68and lRA40t) series and analogues as well as non-ionic crosslinkedpolystyrene polymer resins. for example the Amberlite XAD series oranalogues. preferably XAD-2 and XAD-4. are also suitable to adsorb theantibiotic RlT D-22l4 from the production broth or from an aqueoussolution thereof.

For the recovery of antibiotic RlT D-22l4. systems previously describedfor the extraction of cephalosporin C or penicillin N such as the liquidanionic resins. for example the Amberlite LA series. or extraction intoorganic solvent after N-acylation of antibiotic RlT D- 22l4 in solutionmay be used.

The same techniques those hereinabove mentionned for the recovery ofantibiotic RlT D-22l4 from the fermentation broth are also possibly usedfor its purification. preferably the adsorption technique on weakly orstrongly basic resins or on the non-ionic Crosslinkcd polystyrenepolymer resins. for example. the Amberlite XAD series or the like.Antibiotic RlT D-22l4 may for example be purified by adsorption or crudeantibiotic RlT D-22l4 onto a non-ionic resin of the Amberlite XAD seriesor the like. preferably XAD- 4. followed by elution with deionizedwater.

As indicated hereinabove. antibiotic RlT D-22 l4 may also be obtained bysemisynthetic route. According to this embodiment. D-cysteine is reactedwith a 6- (whaloacetamido)-penicillanic acid (preferably in the form ofa neutral salt such as the sodium, potassium or the ammonium salt). saidb-(a-haloacetamido)- penicillanic acid salt being preferably 6-(01-bromoacetamido )-penicillanic acid ammonium salt according to thefollowing scheme:

2N s "3 i CH N O coon Ha1cn c-na1 with the test organism to determinethe minimum con centration of the ammonium salt of antibiotic RIT D-HaICH CONH coon ti CH HOOC-IEH-CH S-CH C-NH; f 3

N 3 ,OOM

wherein M is hydrogen or a pharmaceutically acceptable nontoxic cationsuch as sodium, potassium and ammonium; Hal is halogen such as chloro,bromo or iodo.

lt is obvious that the semi-synthetic route allows not only theproduction of the D epimer of antibiotic RIT D-22l4 but also theproduction of the L epimer and of the DL racemic form, when startingfrom the L- or DL- cysteine respectively.

2214 in meg/m1. in the broth substrate which inhibited organism growthfor a period of about twenty hours at C.

As indicated in Table l, the monoammonium salt of antibiotic RlT D-22l4exhibits a strong activity against Gram-positive and -negative bacteria.Other salts of antibiotic RlT D-22l4, as for example the calcium saltand the potassium salt exhibit similar antibacterial activity.

TABLE l Minimum Inhibitory Test organism concentration in meg/ml.

The reaction is performed in a suitable solvent such as water at a pHcomprised between 6 and 9, preferably between 7 and 8 at a temperaturecomprised between 0 and C, preferably between 20 and 30 C, according toL. Michaelis and M. P. Schubert in J. Biol. Chem.106, 33l-4l (i934).

Antibiotic RlT D-22l4 exhibits inhibitory activity against the growth ofboth Gram-positive and Gramnegative bacteria.

Antibiotic RIT D-22l4 has been tested in form of the mono ammonium saltin position 3 of the penam nu cleus and, for that reason. is hereinbelow characterized as this monoammonium salt.

The levels at which monoammonium salt of antibiotic RIT D-22 l 4 showsinhibition against the growth of illustrative organisms are set forth inTable l, as a comparative trial with ampicillin. The inhibitory levelswere determined by the broth dilution test. A series of tubes containingvaried concentrations of the monoammo nium salt of antibiotic RlT D-22l4were inoculated Antibiotic RIT D-22l4 exhibits in vivo activity againstGram-positive and Gram-negative microorganisms and, hence, is usefull incontrolling infections caused by such organisms in host animals.However, the efficacy of antibiotic RIT D-22l4 is less advanta geouswhen administered orally.

The level at which monoammonium salt of antibiotic RIT D-22l4 shows 50percent mice protection against infections with illustrativemicroorganisms are set forth in Table 2. Comparative trials withampicillin were performed in vivo according to the usual mice protectiontest procedure. The test compound was administered in graded dilutionsto mice either subcutaneously (so) in water or orally (or) incombination with calcium carbonate one hour after intravenous infectionwith uni formly lethal doses of the test organism. The animals wereobserved for 3 days. The total dose required to protect 50 of theinfected mice is designated as the ED;,.,, the most potent compoundhaving the lowest ED 's.

TABLE 2 Test organism Adminis- RlT D22 14 Ampicillin trationStaphylococcus uureus 663 5.0. l 1.0 [.0 Escherichia cm! 47 so l0.0 23.6Escherichia coli 9 Se I09 I [.5 Salmonella brandebuurg 22 scv [3.0 36.4Salmonella emenlidis 24 s.c. I35 556 Salmonella panama 2U sc. l0.0 22.0Shigella flexneri' 25 S.c. 52.5 lU0.(] Proteus mirabilis l l 5.0. [0.014.8 Klebsiella pneumoniae SK&F 4200 s.c. l H] 36.7 Klebsiellapneumoniae SK&F 4200 or. 25.3 50.4 Vibrio paruhaenmliricux 194 or. 22.7l'l'.9

on a rotary shaker operating with a 50 mm orbit diameter at 150 rpm. Theinoculum so prepared is then utilized in the production of antibioticRIT 02214 as follows into a 20 liter laboratory fermentation tank areadded l5 liters of a production medium (soybean meal 300 g; drieddistiller solubles 75 g; calcium car bonate 90 g; glucose 300 g;saccharose 525 g; L-lysine (50 in water) 30 ml.;L-carboxy-methyl-cysteine 30 Plasma levels after minutes (in meg/ml.)

6.0 3.5 [.0 l7 i2 125 5 23 22.5 l8 18 Mice RlT D-22l4 ampicillin 65 MTD-2214 ampicillin RlT D2214 ampicillin Rats Dog

w! Mb" The monoammonium salt of antibiotic RIT D-22l4 does not show anydetectable toxicity when administered to mice in an acute toxicitytrial. at the relatively high level of 500 mg/kg by intravenous routeand L000 mg/kg by oral route.

The product of this invention may be administered by parenteral or oralroute, being therefore formulated into adequate compositions in the samemanner as other penicillin antibacterials. The dose that is administeredto the subject will depend on the severity and type of infection as wellas the general condition of the subject.

The following examples are presented to illustrate the inventionfurther; they are not to be construed as limiting the scope thereof.

EXAMPLE 1 A sporulated culture of Acremonium chrys'ogenum ATCC 389 isproduced by growing the organism on a nutrient agar slant (soybean meal20 g; calcium carbonate 5 g; glucose 10 g; sucrose 36 g; agar 20 g; oneml. of a 50 L-lysine aqueous solution; deionized water 1 liter) adjustedto pH 7 with phosphoric acid and sterilized for 25 min. at 121 C.

The agar slant is inoculated with spores of Acremuni'um chrysogenum ATCC20 389 and is incubated for 9 days at 25 C. The agar slant is thencovered with 5 to [0 ml. distilled water and gently scraped to removethe spores as an aqueous suspension thereof.

Five ml. of the resulting suspension is used to inoculate 6 liters ofErlenmeyers flasks containing 500 ml. of sterile vegetative medium (cornsteep liquid 10 g; soybean meal g; calcium carbonate 5 g; glucose 20 g;sucrose 20 g; lysine (50 percent in water) 1 ml; de ionized water 1liter) adjusted to pH 7 with phosphoric acid and sterilized for min. at121 C.

The vegetative flasks are shaken for 3 days at 28 C.

g; DLmethionine 60 g; SAG 471 3 ml.; antifoam A 2 ml.; tap water for14.5 liters) at pH 6.5.

After 30 min. sterilization at l2l C, the tank is incubated with 500 ml.vegetative inoculum.

The fermentation is carried out for 4 days at 25 C. aerated with sterileair at a air flow rate in the range of 0.5 to 1.0 volume per volume ofbroth per minute, agitated by a mechanical stirrer equipped with 2 flatblade turbines of 13 cm diameter of 0.48 tank diameter, operating from300 to 450 rpm.

The production of RlT D-22l4 is controlled during the fermentation bysampling at regular intervals and assaying for its antibiotic activityafter adequate dilu tion. by the classical agar diffusion method usingAlca- Iigenes vixculactis as specific test organism.

Oxygen level and pH are continuously recorded during the fermentation inorder to adequately adjust such operating variables as air flow rate,air counterpressure and agitation speed, according to the art. Thefermentation is stopped after a 4 days period. when glucose and sucroseare completely exhausted and the fermentation broth is then supplementedwith 0.1 percent formaldehyde and cooled at 4 C.

Approximately 12 liters of fermentation broth obtained hereinabove areadjusted to pH 3.5 with phosphoric acid and filIfCl The 10 liters brothfiltrate is thoroughly mixed for 45 min. with 300 g activated car bon(NORlT D sold by Norit Sales Corporation Ltd. Amsterdam. TheNetherlands), the pH being adjusted to 2.5 with hydrochloric acid. Thecharcoal is filtered off on Dicalite (a diatomaceous earth sold byJohns-Manville Products Co. New York. USA). washed with water up todecoloration of the effluent and then eluted with a 60:40 acetone/watermixture.

The eluate is concentrated under reduced pressure to remove acetone andpoured at a flow rate of 500 ml./h onto a 8 X 42 cm column packed withlRA-402 resin 13 (formate cycle) (an anion exchange resin sold by Rohm &Haas Co., Philadelphia, USA) and subsequently washed with formic acid toconvert it to the formate cycle and with deionized water to reachneutral pH.

The column is washed with water until the effluent is clear andcolorless and the antibiotic removed by eluting with 7.5 liters of a 0.2M ammonium formate solution, at a flow rate of l l./h. Theantibiotically active fractions (3.6 l.) are combined and concentratedunder reduced pressure to approximately [40 ml.

The concentrate is then passed at a rate of 100 ml./h over a 4 X 60 cmcolumn packed with XAD-2 resin (:1 macro-reticular nonionic polymer soldby Rohm & Haas Co., Philadelphia, USA and previously washed withdeionized water up to neutral pH and low conductivity level). Theantibiotic is fixed and eluted with deionized water at a rate of 200ml./h. The most active fractions are combined and concentrated underreduced pressure by azeotropic distillation with isopropanol. The crudeproduct is recovered as a yellow powder by treating the syrupyconcentration with hot ethanol, evaporating the solvent under reducedpressure and washing the precipitate with ether to yield crudemonoammonium salt of RlT D-22l4.

EXAMPLE 2 Five hundred milligrams aliquot of the crude antibiotic RITD-22l4 obtained at the end of example 1, are dissolved in 2 ml. ofdeionized water and the solution is poured onto a 1.0 X 30 cm columnpacked with XAD 4 resin (a nonionic macroreticular polymer sold by Rohm& Haas Co., Philadelphia, USA) and previously washed with deionizedwater up to neutral pH and low conductivity level. The antibiotic RlTD-22l4 is eluted by washing the column with deionized water. The mostactive fractions are combined and concentrated under reduced pressure byazeotropic distillation with isopropanol. The monoammonium salt ofantibiotic RIT D- 2214 is obtained as a white powder by treating thesyrupy concentrate with hot ethanol, filtering and washing theprecipitate with ether.

EXAMPLE 3 Approximately 12 liters of fermentation broth, obtainedaccording to the procedure described in the first part of example 1, aresupplemented with 0.1 percent formaldehyde and filtered on Dicalite (adiatomaceous earth sold by Johns Manville Products Co.). The filtratel.) is adjusted to pH 5 with phosphoric acid and passed over a 4 X 7.5cm column of IR 45 (OH') resin (a weakly basic anion exchange resin soldby Rohm & Haas Co., Philadelphia, USA), subsequently washed with Nsodium hydroxyde to convert the resin to the basic form and withdeionized water until pH is below 9.0, at a flow rate of4 l./h. Thepercolate is acidified to pH 2.5 by slow addition of IR 124 (H*) resin(a strongly acid cationic exchange resin sold by Rohm & Haas Co.,Philadelphia, USA), subsequently washed with N sulfuric acid to convertit to the acid form and with deionized water until pH is above 4 andpoured through a 5 X 35 cm column packed with IRA 68 resin (formatecycle, as in example 1), at a rate of 500 ml./h. The column is washedwith deionized water until the effluent is clear and colorless and theantibiotic is eluted with 3.5 l. 0.2 M ammonium formate, at l l./h. Theantibiotically most active fractions (1 l.) are collected andconcentrated under reduced pressure to approximately 100 ml. and pouredonto a 4 X 40 cm column packed with XAD 2 resin prepared as indicated inexample 1. The elution is performed by washing with 5 200 ml./hdeionized water.

Crude ammonium salt of antibiotic RIT D-2214 is recovered from the mostactive fraction of the eluate ac cording to the isolation proceduredescribed in example 1.

EXAMPLE 4 A l g aliquot of crude ammonium salt of antibiotic RIT D-22l4obtained at the end of example 3 is dissolved in 2.5 ml. ofwater and 5ml. of isopropyl alcohol is added thereto with stirring. The solution ispoured in a polyamide cylinder (4.5 X 50 cm) packed with microcrystalline cellulose and thereafter developped with a 70:30 mixture ofisopropyl alcohol and water. The col umn is then sliced (according tothe dry-column chromatography method described by B. Loev and M. M.Goodman in Progress Separation Purification vol. 3, p. 7395, 1970) in 5cm thick segments which are poured in sintered glass funnels andtriturated with a 50 ml. portion of water and the slices are checked forpresence of antibiotic by microbiological testing using Alcaligenesviscolactis as test organism.

The pooled solution is treated by azeotropic distillation in thepresence of isopropyl alcohol to yield a concentrated solution fromwhich crystallization occurs at 4 C. The precipitate is filtered, washedwith cold ethanol and dried over phosphorous pentoxide for 3 days at 25C under reduced pressure to yield the ammonium salt of antibiotic RITD-22l4 as a white, solid, decomposing at about 142 C, very soluble inwater, slightly soluble in methanol and insoluble in other organicsolvents.

Analysis calculated ("/01 found (7%) l irt m i a zl C 39.58 39.52 H 5.625.65 N I420 14.26

The D configuration of antibiotic RlT D-22I4 was demonstrated bydetermination of the D-configuration of its carboxymethylcysteinefragment. Therefore, the ammonium salt of antibiotic RIT D-22l4 washydrolyzed in normal hydrochloric acid for 8 hrs. at 100 C undernitrogen atmosphere and the obtained carboxymethylcysteine was isolatedby column chromatography on strong basic anionic resin. The mixedmelting point between this product and D-carboxymethylcysteine obtainedby chemical synthesis is l85-186 C, i.e. identical to the melting pointof the chemically synthesized product, the melting point of thechemically synthesized L-carboxymethylcysteine being l95196 C.

The infrared absorption spectrum of the monoammonium salt of antibioticRlT D-22l4 in l potassium bromide presents distinguishable bands in theinfrared spectrum over the range of 1,000 to 4,000 cm as follows 3,400(broad band); 2,950; 1,770; l,650l,600 (broad band), L540 (shoulder);1,410; 1,330; 1,l20.

The NMR spectrum of antibiotic RIT D-22l4 at l5 percent in D 0. withtetramethyl silane as reference, shows the following characteristics 8.4ppm(d); 6.85

ppm(t); 6.6 ppm(s); 6| ppm(q); 5.85 ppm(s); 45 ppm(q).

Paper chromatography on Whatman No. 4 paper of the monoammonium salt ofantibiotic RIT D-22l4 gives the following Rf values in two differentsolvent systems (comparison made with ampieillin).

Solvent system (upper phase) Ant. RIT D 22l4 Ampicillin nButanol.ethanol, water (HR 0.43

mButanol, acetic acid. water 043 0.69

nbutanol, ethanol. acetic acid. 072 water l(J:l.5:l.5:2)

n-hulanol. acetic acid. water (4:1: l) 0.63 acetonitrilc. wutcr (75:25)v 0.22

Antibiotic RIT D-22 l 4 shows a positive test with ninhydrin. sodiumhydroxyde-iodine, sodium azide and chloroplatinic acid. Antibiotic RlTD-22l4 can be colori metrically assayed by the classical hydroxylaminemethod of penicillin assay. Antibiotic RIT D-22l4 is relatively stableunder acid conditions. Comparative trials were performed with themonoammonium salt of antibiotic RlT D-22l4. penicillin G and ampicillin,in reconstituted gastric juice, at pH [3. Recoveries were estimatedaccording to the hydroxylamine assay method. The correspondinghalf-life, expressed in minutes, are respectively 7 for penicillin G, 25for antibiotic RIT D-2214 and 390 for ampicillin. Antibiotic RlT D-22 l4is relatively stable when kept at 4 C in a water solution for not morethan 7 days.

Antibiotic RIT D-22l4 is rapidly destroyed by a Staphylococcalpreparation of B-lactamase (Penase Leo from Leo pharmaceuticalsproducts, Ballerup, Danemark).

EXAMPLE 5 Twelve liters of fermentation broth obtained accord ing to theprocedure described in the first part of Examale 1 are extractedaccording to the procedure of Example 3, but the elution of the IRA 68resin is performed with 3.5 l. of 0.2 M sodium acetate. The most activefractions are collected and treated as described it Example 3 to yieldsodium salt of antibiotic RIT D- 22 l4.

EXAMPLE 6 A sporulated culture of Acremrmium chrysogenum ATCC 20 389 isproduced by growing the organism on i nutrient agar slant (soybean meal20 g; calcium car- )onate 5 g; glucose 10 g; sucrose 36 g; one ml. of 50percent L-lysine aqueous solution; agar 20 g; deionized water 1 liter)adjusted to pH 7 with phosphoric acid and sterilized for 25 min. at 121C.

The agar slant is inoculated with spores of Acremonr'um chrysogenum ATCC20 389 and is incubated for 9 days at 25 C. The agar slant is thencovered with 5 to l0 ml. distilled water and gently scraped to removethe spores as an aqueous suspension thereof.

One ml. of the resulting suspension is used to inoculate 250 ml.Erlenmeyers flasks containing 500 ml. of sterile vegetative medium (cornsteep liquid l0 g; soybean meal 30 g; calcium carbonate 5 g; glucose 20g; sucrose 20 g; one ml. of 50 percent L-lysine aqueous solution;deionized water 1 liter) adjusted to pH 7 with phosphoric acid andsterilized for 35 min. at 121 C.

The vegetative flasks are shaken for 3days at 28 C. on a rotary shakeroperating with a 50 mm orbit diame' ter at 260 rpm. This inoculum isthen used in the production of antibiotic RIT D-22l4. A production medium consisting of: soybean meal l5g; calcium carbonate Sg; glucose 10g; saccharose 36 g; DL-methionine l g; L-lysine (50 percent in water) 1ml.; L-carboxymethylcysteine 0.5 g; deionized water I liter, adjusted topH 7 with phosphoric acid and distributed in 250 ml. Erlenmeyers flaskswith 30 ml. medium content and sterilized for 35 min. at 121 C isinoculated with 5 percent vegetative inoculum and incubated at 25 C for5 days on a rotary shaker operating with a 50 mm orbit diameter at 260rpm.

The production of antibiotic RIT D-22l4 is controlled during thefermentation by sampling at regular intervals and assaying for itsantibiotic activity after adequate dilution by a classical agardiffusion method using Alcaligenes viscolacris as specific testorganism.

EXAMPLE 7 Using the technique of example 6 but replacing the thereindescribed production medium by a production medium having the followingcomposition (soybean meal 15 g; dried distiller solubles 5 g; glucose [0g; mo- [asses 20 g; DL-methionine 2 g; L-lysine (50 percent in water) lml; L-carboxymethylcysteine 0.5 g; deionized water 1 liter) adjusted topH 7 with phosphoric acid, crude antibiotic RlT D-22 I4 is obtained.

EXAMPLE 8 Using the technique of example 6 but replacing the thereindescribed production medium by a production medium having the followingcomposition herring meal 20 g; calcium carbonate 3 g; glucose l0 g;saccharose 40 g; oleic acid 5 ml.; D-L methionine l g; one ml. of 50percent L-lysine aqueous solution; L-carboxymethylcysteine 0.5 g;deionized water 1 liter) adjusted to pH 7 with phosphoric acid, crudeantibiotic RlT D- 2214 is obtained.

EXAMPLE 9 To a solution of 3.63 g (0.03 mole) of D-cysteine and 2.4 g(0.03 mole) of ammonium bicarbonate in ml. of water flushed withnitrogen, there is added, at once, with vigorous stirring ll.25 g (0.03mole) of 6-(crbromoacetamido)-penicillanic acid ammonium salt (preparedaccording to Y. G. Perron. W. F. Minor et al, in J. Amer. Chem. Soc. 82,39348, 1960).

The mixture is stirred at room temperature for about 30 minutes. thenrapidly cooled to 5 C, layered with 3 X l00 ml. of methyl isobutylketone(MlBK) and ex- 17 18 tracted by mixing vigorously while adjusting the pHto The activity of this compound against Gram-positive 3 with DOWEX SOXl2(H )(a product of Dow Chem. and negative bacteria has approximatelyhalf the value 10-. Midland Michigan. of that shown by the RIT L-22l4.

The combined MIBK extracts are eliminated and the aqueous phase isconcentrated up to 20 ml. by evapora- EXAMPLE 13 tion under reducedpressure at 30 C, the pH being ad- Using the procedure described inexample 9 but rejusted to 7 by the addition of a few drops of 25 percentla ing 6 (a-br0moacetamid0)-penicillanic acid amammonium hydroxidesolution. The residue is taken up monium lt by6-(a-bromoacetamido)-penicil|anic in 500 ml. ice-cold ethanol. stirredfor 20 minutes and id sgdium alt re ared a ording to Y. O. Permn, thethus obtained precipitate is filtered, washed with 0 w F, Mi r L i J,Amer. Chem. Soc, 82, 393443 ml. cold ethanol and 50 ml. diethyl etherand dried over [960), crude sodium salt of antibiotic RIT D-22 l 4 is 25 under reduced Pressure to yield the crude ammo obtained. This crudeproduct is purified as indicated in nium salt of antibiotic RIT D-22 l4.examplg 10,

EXAMPLE l0 [5 EXAMPLE l4 Two grams of crude amm nium a of afltibiolifiRIT An injectable pharmaceutical composition is formed D-22l4 Obtainedat he end Of example 9 1H i s lVfi by adding sterile water or sterilesaline solution (2 ml.) in 5 ml. of water and poured in a column (4 X 75cm) to 500 mg f R|T 112214 (monoammonium l y packed with 628 ml. ofXAD-4 resin in water. The column is eluted with water, each 25 ml.fraction collected EXAMPLE being checked up for antibiotic content byconductivity A ib r im capsme h h f n i compoand thin-layerchromatography. nents RIT D-22l4 (monosodium salt) 500 mg; cal Theso-obtained fractions are combined, treated in i Carbonate 250 magncsiumte-grate 75 the same as in the beginning of example 4 and freezew l idried to yield pure ammonium salt of antibiotic RIT D- 25 l, 6-(D)-(l2-amino-2-carboxy)-ethylthio]- 2214 showing the same physico-chemicalcharacterisr id i illani a id f th f l tics as those described at theend of example 4.

EXAMPLE ll (1 llUUC 5 CH. Using the procedure described in the example 9but \VTH :HA S CH replacing D-cysteine by L-cysteine, crude ammoniumFiji l i salt of antibiotic RIT L-22l4 is obtained. This crude @3 1product is purified as indicated in the example 10.

The following Table 4 shows that the monoammonium salt of antibiotic RITL-22l4 also is active against or a salt thereof with a pharmaceuticallyacceptable Gram-positive and negative bacteria, but less than thecation. monoammonium salt of RIT D-22l4. 2. A compound according toclaim 1 which is a salt TABLE 4 Staphylococcus aureus L25 1.25Slaphylococcus uureus Pcn. res. 80.0 20.0 Escherichia cull 40.0 1.25Escherichia Coll Pen. resistant 80.0 80.0 Aerubacler aerogenei' 20.0 l.25 Klebsiella pneumoniae 80.0 40.0 Salmonella paratyphi 20.0 [.25Shigella .wnnei 80.0 2.5 Pmieus mimbilir 20.0 1.25 Proteus mnrganii 80.020.0 Pseudumonas aeruginosu 80.0 80.0 Bacillus subrilix 0.3l 0.3

EXAMPLE 12 wherein the salt 15 a mono-salt with a pharmaceuticallyacceptable cation.

Using the procedure described in example 9 but re 3 A Compound accordingto claim 2 wherein the placing DL-cysteine. crude ammonium salt ofantibi- I otic RIT DL22l4 is obtained. This crude product ispharmaceutically acceptable Canon is Sodmm potas purified as indicatedin the example 10. slum or immomum;

1. 6-(D)- (2-AMINO-2-CARBOXY)-ETHYLTHIO)-ACETAMIDO PENICILLANIC ACID OFTHE FORMULA:
 2. A compound according to claim 1 which is a salt whereinthe salt is a mono-salt with a pharmaceutically acceptable cation.
 3. Acompound according to claim 2 wherein the pharmaceutically acceptablecation is sodium, potassium or ammonium.